Diphtheria toxin (DT) and pseudomonas exotoxin A (PE) are potent cytotoxic proteins produced by Corynebacterium diphtheriae and Pseudomonas aeruginosa. Both toxins are virulence factors for the organisms secreting them, as well as serving as useful probes in Cell Biology. Both proteins exert their toxic effect by stopping protein synthesis in mammalian cells; both are enzymes and act by ADP ribosylation of cytoplasmic elongation factor 2. The structures of these two enzymatically identical proteins however are different. In order to exert a biological effect on sensitive cells, both toxins bind to specific cell surface receptors, are internalized via coated areas into endosomes, delivered to the Golgi region and then to lysosomes. During this intracellular trafficking the toxins are converted to an enzyme active form, presumably in endosomes or Golgi associated vesicles. The site of escape into the cytoplasm is unknown. In the next grant period, we propose to: (1) Definitively define the sites and mechanisms for processing both PE and DT in sensitive cells, and determine if they are similar or different in resistant cells. This understanding will allow us to determine if the manner in which a mammalian cell handles a toxin dictates the cell's susceptibility to that toxin. (2) Isolate and characterize the receptor for PE on mouse LM fibroblasts. Once the receptor has been purified, partially sequenced, and antibodies made to it, the cDNA for the receptor can be cloned. (3) Define the intracellular pathway for the PE receptor in LM cells. We will determine if receptor follows the same pathway in the presence and absence of PE, and the site of toxin-receptor dissociation. Experiments on toxin trafficking and processing involve subcellular fractionation and identification of toxin by ELISA, or Induction of ADP ribosyl-transferase activity with gradient fractions. Toxin movement on the ultrastructural level will be followed using biotinyl-toxin-streptavidin-gold, or by pre-embedding immunochemical techniques. Receptor movement will be characterized both with electron microscopy and with biochemical probes.